The Mycobacterium genus comprises over 50 species, including major pathogens for the human such as Mycobacterium tuberculosis, agent of tuberculosis, Mycobacterium leprae, agent of leprosy, and Mycobacterium avium, a major opportunist in the course of AIDS. Little is known of the genetic mechanisms of the virulence of M. tuberculosis and the other pathogenic species for the human, due to the low growth rate of these bacteria and the lack of adequate cloning vectors, and as a result it is difficult to insert foreign DNA in these microorganisms. Moreover, the mycobacteria show a particular resistance to many antibiotics due to the fact that the constitution of their wall is poorly permeable to these molecules. However, recent studies have shown that it is possible to introduce foreign DNA in BCG with a view to expressing antigens heterologous to this bacterium.
In the case of BCG manipulation, the use of markers of resistance to antibiotics hitherto available in mycobacteria such as the aph3 gene providing a high degree of resistance to kanamycin or genes providing resistance to streptomycin or spectinomycin, cannot be envisaged because it could cause a dissemination of resistance to these antibiotics in the environment in the event that recombinant BCG is used for therapeutic or prophylactic purposes in the human or the animal. It is thus of the utmost importance to be able to develop resistance markers which are harmless for the environment in order to select recombinant mycobacteria which can be widely used.
At the present time, only a very small number of markers usable in mycobacteria is available.
Thus, Aldovini et al. (J. Bacteriol, 1993, 175, 7282-7289) have isolated the gene coding for orotidine-5'-monophosphate decarboxylase (OMP-DCase) of Mycobacterium bovis BCG.
Another marker, the cI gene, coding for the L1 phage repressor, has also been used (patent application WO- 90/00594).
Shuttle vectors based on these markers and allowing the transfer of DNA between Escherichia coli and Mycobacterium have already been described. Thus, patent application WO-91/13 157 describes two shuttle vectors, pEP2 and pEP3, respectively carriers of a gene of resistance to kanamycin and hygromycin.
One of these vectors, pEP2, is modified by the introduction of the protein promoter MBP70 of Mycobacterium, as described in patent application WO-90/10 701.
Other shuttle vectors usable in mycobacteria and carrying markers of resistance to antibiotics, auxotrophic markers or the cI gene have been described in patent application WO 90/00594.
Finally, Ranes et al. (J. Bacteriol. 1990, 172, 2793-2797) have described the construction of a shuttle vector called pRR3 carrying a gene of resistance to kanamycin expressing itself in Mycobacterium bovis BCG and Mycobacterium smegmatis.
A strain of non-pathogenic saprophytic mycobacterium resistant to mercury carrying a plasmid of 115 MD was also known (Meissner and Falkinham, J. Bacteriol, 157, 669-672, 1984). This very large plasmid was not, however, characterized. In particular, the genes involved in the resistance to mercury were neither localized nor identified, and consequently this plasmid could not be used as a shuttle plasmid. Moreover, saprophytic mycobacteria do not belong to pathogenic mycobacteria, the latter being principally included in the M. tuberculosis, M. bovis and M. leprae species.
The result was that those skilled in the art wishing to transfer DNA coding for antigens, obtained by cloning in Escherichia coli, to mycobacteria with a view to fabricating vaccines, was confronted with a lack of easy-to-manipulate vectors carrying markers expressing themselves in E. coli and mycobacteria, and without danger to human or animal health. The use of genes of resistance to antibiotics of therapeutic aim must, in fact, be ruled out for the reasons given above. The use of auxotrophic mutants gives rise to practical difficulties of selection and moreover involves the need to obtain mutants for each mycobacterium strain.